We have uncovered a unique system for analysis of glucocorticoid receptor (GR) recycling. In addition to providing new Insights into nucleocytoplasmic shuttling of this signal transduction protein, our results implicate protein phosphatases as potential mediators of transformation by v-mos oncoproteins. Our immediate plans are to continue utilizing biochemical, genetic, and molecular and cell biological approaches to study the effects of v-mos oncoproteins on regulated nuclear transport of GRs. In addition, we plan more detailed investigations into the role of specific protein phosphatases in v-mos transformation. In particular, we will examine the phosphorylation state of GRs by 2-dimensional mapping of phosphotryptic peptides. The effects of okadaic acid, a specific protein phosphatase type 1 and 2A inhibitor, on recycling and phosphorylation of GRs in vivo will be examined by indirect immunofluorescence and 2-dimensional tryptic mapping, respectively. We also plan to examine whether other cellular factors associated with GRs In multiprotein complexes influence their recycling. Finally, we will use a novel genetic selection scheme to screen for potential GR, v-mos oncoprotein, protein phosphatase, and cellular mutations that after GR recycling. Many of our planned experiments involve collaborative efforts with other investigators who offer areas of expertise not available in my current environment. We plan to perform biochemical analysis of protein phosphatases in the laboratory of Dr. Brautigan (Brown University). We hope to uncover, at the molecular level, the nature of the interaction between v-mos oncoproteins and protein phosphatases. The direct role of protein phosphatases In regulating glucocorticoid receptor recycling in vivo will be addressed using microinjection experiments to be performed in the laboratory of Dr. Ned Lamb (CNRS, Montpillier, France). Finally, in order to facilitate the development of an in vivo nuclear export system, I plan to spend some time In the laboratory of Dr. Douglass Forbes (University of California, San Diego). The ability to expand my research program into these novel and unique areas is somewhat hampered by my teaching and departmental duties which currently consume 50% of my time each academic year. With an RCDA my departmental and university duties will be significantly reduced (10% effort/ academic year), and thus will have the freedom and time that will be required to gain the valuable experience in the laboratories of my collaborators. In each case, I expect to transfer the technologies learned to my own laboratory. My long term research goals would also be greatly facilitated by an , since my future experiments would not be limited to the expertise and technology that exists In the laboratory. I expect to continue in the future collaborations that have already been established so that my research program can continually take advantage of technologies developed by current and future collaborators.